Recently, significant attention has been paid to the cysteine knot protein (CKP) family due to their potent biological effects and modulation of their functions appears to be a promising therapeutic approach. By a bioinformatics approach, we have identified a novel member of CKP family, "Vwc2 (von Willebrand factor C domain containing 2)", and explored its potential functions. Our preliminary data indicated that Vwc2 gene expression is associated with early osteoblast differentiation, and that the Vwc2 protein is present in bone matrix and preferentially localized at the bone surface. When Vwc2 was stably overexpressed in MC3T3-E1 (MC) osteoblastic cell line, in vitro mineralization was markedly inhibited possibly via its specific binding to a transforming growth factor (TGF)-ss superfamily member, 2A subunit isoform of activin A. Moreover, when Vwc2 was added together with activin A protein, Smad2 phosphorylation was synergistically enhanced compared to either activin A or Vwc2 addition alone in MC cells indicating that Vwc2 facilitates activin signaling. Recently, several lines of evidence suggests that activin signaling inhibits osteoblastic differentiation and mineralization, whereas its inhibition of this signaling pathway leads to enhance bone formation and bone strength in vitro and in vivo. As a preliminary step of obtaining such objectives, we have just generated mice, in which the Vwc2 gene has been selectively overexpressed in tissues of interest. Thus, we hypothesize that a novel CKP member, Vwc2, modulates mineralization in bone by assisting activin signaling in osteoblasts. The objectives of this exploratory proposal are to establish the function of Vwc2 in activin signaling in vitro and to determine the effects of Vwc2 modulation on biomineralization in vivo. In order to test the hypothesis, the following specific aims are proposed. 1. To investigate the potential signaling pathway by which Vwc2 modulates osteoblast function in vitro. 2. To investigate roles of Vwc2 in bone formation in vivo. The data obtained from this study may provide insights into the biological functions of this novel CKP member in bone formation and help a new molecular design for therapies of bone formation and healing. PUBLIC HEALTH RELEVANCE: Although current osteoporosis drug market is heavily dominated by anti-resorptive agents, they fail to promote the replacement of bone loss. Our studies not only advance our knowledge of a new CKP member, Vwc2 in Activin signaling but also help define the molecular mechanisms of this signaling pathway leading to the inhibition of bone formation. By accomplishing our studies proposed, the logical extension of this research will further allow controlling the Vwc2 function in osteoblasts, thereby allowing the potential anabolic effects on bone formation, as well as provide new knowledge of bone formation critical for efforts directed at decreasing the risk of bone fracture.